The diplococcic bacterium Neisseria gonorrhoeae (GC) is the causative agent of the sexually transmitted infection (STI) gonorrhea. Annually, gonorrhea affects over 78 million individuals worldwide. While the overall incidence has been decreasing, the emergence of multiple cases of antibiotic resistant isolates has again made gonorrhea a focus for intervention. In women, up to 50% of gonorrhea cases may be asymptomatic meaning that the infection remains untreated. This untreated infection can have serious reproductive health consequences with the potential development of pelvic inflammatory disease (PID), which in turn can lead to serious sequelae such as ectopic pregnancies and infertility. Phase variation in GC has been observed as early as the 1970?s when clinical isolates grown on agar plates were classified into four groups based on the presence or absence of pilin and Opa proteins. While research on the effect of variation on infectivity is ongoing, characterization of the various surface proteins expressed during different stages of infection has not been studied. For the purpose of this proposal, enhanced infectivity or more infectious refers to isolates recovered from the female upper genital tract, i.e. the endometrium, uterus or fallopian tubes, and/or exhibit more severe symptomatology, including abdominal pain which may indicate complicated gonococcal infections such as PID. The ability of major surface proteins to vary their expression significantly complicates the development of a vaccine against GC. Recent advances in vaccine development that incorporate bioinformatics analysis to select potential vaccine candidates have emerged. Reverse vaccinology, pioneered by Dr. Tettelin in collaboration with the group of Dr. Rappuoli (Chiron, Novartis Vaccines, GlaxoSmithKline), was first used towards a protein-based vaccine against the other pathogenic member of the Neisseria genus, N. meningitidis. This led to the development and successful implementation of the 4CMenB vaccine (Bexsero) and provided renewed optimism that a N. gonorrhoeae vaccine is attainable. A 2016 proteomics-based study by Zielke et al. provides additional evidence of the feasibility of this type of analysis in our search for gonococcal vaccine targets. More detailed analysis, facilitating a more focused and streamlined process, can then be implemented to test the best vaccine candidates. Our project builds upon these foundations and focuses on GC changes that occur under relevant physiological conditions that would be encountered in vivo. We hypothesize that specific differentially regulated GC surface proteins are selected for during infection and these events enhance infectivity across multiple strains, thus providing targets for vaccine development. We will first characterize these profiles using GC laboratory strains then extrapolate them to similarities in profiles observed between GC clinical isolates that are readily transmitted and/or display enhanced infectivity.